Volume 13, Issue 8, Pages 674-679 (April 2003) The prickle-Related Gene in Vertebrates Is Essential for Gastrulation Cell Movements  Masaki Takeuchi, Jun Nakabayashi, Takuya Sakaguchi, Takamasa S. Yamamoto, Hiroki Takahashi, Hiroyuki Takeda, Naoto Ueno  Current Biology  Volume 13, Issue 8, Pages 674-679 (April 2003) DOI: 10.1016/S0960-9822(03)00245-8

Figure 1 Xpk and Zpk Antisense Morpholino Oligos (MO) Inhibit Convergent Extension Movements (A) Whole-mount in situ hybridization of Xpk in staged embryos; early gastrula stage 10.5 (a and b, vegetal and lateral views, respectively), late gastrula stage 13 (c and d, vegetal and dorsal views, respectively), and early neurula stage 15 (e and f, posterior and dorsal views, respectively). In (a), (c), and (e), the dorsal side is at the top. In (b), the dorsal side is at the right. (B) Northern blot analysis of Xpk. The numbers at the top of each lane indicate developmental stages. Two Xpk transcripts of around 5 kb are detected in eggs to stage 32 embryos. ODC is the RNA loading quantity control. (C) Animal cap assay; Xpk expression is induced in animal cap cells in a dose-dependent manner by the overexpression of Xbra. (D) mo-Xpk injection perturbs the correct gastrulation movements and activin-induced elongation of animal cap explants. (a) Uninjected sibling embryo at the tadpole stage (left panel) and 0.5 pg activin mRNA-injected animal cap explants (right panel). (b) Unrelated mo has no effect when injected dorsally (left panel) or in the animal cap elongation assay (right panel). (c) 4mis mo-Xpk has little effect when injected dorsally (left panel) or in the animal cap elongation assay (right panel). (d) mo-Xpk 10-pmol dorsally injected embryo (left panel) and animal cap coinjected with activin and mo-Xpk (left panel). (E) mo-Xpk prevented the translation of overexpressed Xpk-GFP, but unrelated mo or 4mis mo-Xpk had no or little effects, respectively. Translation of coinjected GFP mRNA as a negative control was not prevented. (F) In the animal cap elongation assay, mo-Xpk does not interfere with mesoderm induction (Xbra expression) or expression of either Xwnt11 or Xpk itself. Histone H4 is the cDNA loading quantity control. (G) Zpk knockdown with mo-Zpk inhibits dorsal convergence. Phenotypes are observed at the10 somite stage, 17 hpf ([a and b], lateral view; [c–f], dorsal view; [g and h], anterior view) and prim-20 stage, 33 hpf (i–k). (a, c, e, g, and i), uninjected controls. (b, d, f, h, and j), mo-Zpk 0.5 pmol-injected phenotypes. These phenotypes were similar to mo-Zpk version 2 that recognizes the distinctive site of Zpk 5′-UTR (our unpublished data). (k) 4mis mo-Zpk-injected phenotype. (e,f) Whole-mount in situ hybridization of myoD (red). Current Biology 2003 13, 674-679DOI: (10.1016/S0960-9822(03)00245-8)

Figure 2 Structure-Function Relationship of XPK: Overexpression of Wild-Type and XPK Deletion Constructs (A) Structures of wild-type XPK and deletion constructs; ΔPET, ΔLIM, ΔP/L, and P/L are illustrated in (a). (b) All of these constructs inhibit activin-induced elongation of animal cap explants (wild-type and ΔPET, 500 pg; ΔLIM, 1 ng; ΔP/L and P/L, 250 pg). (c) None of the XPK constructs affect activin-induced Xbra and Xwnt11 expression in animal cap explants at stage 10.5. The indicated numbers correspond to (b1–6). (B) All of the constructs perturb normal gastrulation movements when injected into the dorsal embryo. Phenotypes of abnormal gastrulation were classified into three grades that indicated (a) (1) a normal or weak phenotype showing a small head but nearly normal-sized trunk and tail, (2) an intermediate phenotype showing a short and curved body axis, or (3) a severe, spina bifida-like phenotype showing an open blastopore. (b) Effects of LIM domain-deleted Xpk injection are rescued by the coinjection of wild-type Xpk mRNA. In this bar graph, the phenotypes caused by injection of these constructs into the dorsal embryo were scored as 1, 2, or 3 and are indicated with white, gray, and black color, respectively. (c) An example of phenotype rescue. The result of injecting ΔP/L at 250 pg is shown in the upper panel, and the phenotype resulting from injecting ΔP/L with wild-type Xpk at 500 pg is shown in the lower panel. The ratio of abnormal embryos was reduced by the coexpression of wild-type Xpk. Current Biology 2003 13, 674-679DOI: (10.1016/S0960-9822(03)00245-8)

Figure 3 XPK Interacts Physically with Dishevelled and JNK to Activate JNK in HEK293T Cells (A) JNK activation could not be detected in Xpk-transfected cells, but Xdsh alone could activate JNK in a dose-dependent manner. (B and C) Two-phase XPK activity, depending on the Xdsh dose. (B) At a low Xdsh dose, wild-type XPK enhances Xdsh-mediated JNK activation. (C) At a high Xdsh dose, wild-type XPK or ΔP/L suppresses the activation of JNK, which is mediated by Xdsh. HEK293T cells were transfected with the indicated expression plasmids. The activation of JNK was monitored with an anti-phosphorylated c-Jun antibody. Western blotting with an anti-Gal4DBD antibody was the control for the transfection and loading quantity. (D) XPK interacts physically with Xdsh. (E) Wild-type XPK interacts physically with JNK1. Current Biology 2003 13, 674-679DOI: (10.1016/S0960-9822(03)00245-8)